RESUMEN
Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL(1) particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-ß-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts.
Asunto(s)
Endocitosis/fisiología , Leishmania mexicana/metabolismo , Lipoproteínas LDL/metabolismo , Microdominios de Membrana/metabolismo , Receptores de LDL/metabolismo , Animales , Bovinos , Ésteres del Colesterol/metabolismo , Esterificación , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Humanos , Leishmania mexicana/efectos de los fármacos , Leishmania mexicana/crecimiento & desarrollo , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/sangre , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , beta-Ciclodextrinas/farmacologíaRESUMEN
BACKGROUND: Reservosomes are lysosome-related organelles found in Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells. METHODOLOGY/PRINCIPAL FINDINGS: Using Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones. CONCLUSIONS/SIGNIFICANCE: Taken together, our results suggest that epimastigote forms of T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation.
Asunto(s)
Colesterol/metabolismo , Cuerpos de Inclusión/metabolismo , Estadios del Ciclo de Vida , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Fluorometría , Cuerpos de Inclusión/efectos de los fármacos , Cuerpos de Inclusión/ultraestructura , Estadios del Ciclo de Vida/efectos de los fármacos , Microscopía Fluorescente , Oxazinas/metabolismo , Factores de Tiempo , Trypanosoma cruzi/citología , Trypanosoma cruzi/ultraestructuraRESUMEN
CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells.